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Hypoglycemic effect of Cecropia peltata L. on N5-STZ type 2 diabetic rats

Article  in  Pharmacologyonline · January 2007

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Pharmacologyonline 3: 203-210 (2007) Andrade-Cetto et al.

HYPOGLYCEMIC EFFECT OF CECROPIA PELTATA L.

ON N5-STZ TYPE 2 DIABETIC RATS

Adolfo Andrade-Cetto a* René Cárdenas b and Brenda Ramírez-Reyes a

a
Lab. Etnofarmacología, Fac. de Ciencias, Universidad Nacional Autónoma de
México, México DF.
a
Lab.Biologìa Animal Exp., Fac. de Ciencias, Universidad Nacional Autónoma de
México, México DF.

*Corresponding author: aac@fciencias.unam.mx

Summary

Cecropia peltata is a plant highly appreciated in the Mayan communities of the south west
part of México for the treatment of type 2 diabetes. In this work the hypoglycemic effect of
the aqueous (Ae-P) and the butanolic (Be-P) extracts of the plant were tested in n5-stz
diabetic rats. F The Ae-P showed a hypoglycemic effect only at 200 mg/kg bw after 180
min of administration, whereas 20 mg/kg bw did not show any effect. The Be-P at 27
mg/kg bw showed effect only at 180 min., whereas at 60 mg/kg bw showed an
hypoglycemic effect since 60 min up to 180 min. Since C. peltata extracts exerted a lower
hypoglycemic effect than that previously observed with Cecropia obtusifolia, after we
compared the phytochemical profile of both species we observe that C. peltata presents
lower concentrations of Cholorogenic acid, with this results one can assume that a good
hypoglycemic effect of both Cecropia species is related with the amount of Cholorogenic
acid and Isoorientin present in the extracts.

Key Words: Neonatal induced diabetic rats, medicinal plants, type 2 diabetes, Cecropia.

Introduction

Type 2 diabetes is one of the primary threats to human health, due to its increasing
prevalence, chronic course and disabling complications. The natural history of type 2
diabetes begins with a period of insulin resistance, with augmented pancreatic insulin
secretion. As the disease progresses, pancreatic function falters and is no longer able to
meet peripheral demands. As a result, insulin levels fail to keep up with the body
requirements. The disease is characterized by increased circulating glucose concentrations,
associated with abnormalities in carbohydrate, fat and protein metabolism and a variety of
microvascular, macrovascular, neurologic and infectious complications (1).

In Mexico about 10.6% of the population between 20 and 69 years old is affected by the
disease, ranging around the ninth place worldwide (FMD, 2007). The use of medicinal

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plants among the Mexican population is a tradition. The use of 306 species for the
treatment of type 2 diabetes has been already reported (2). Among them Cecropia
obtusifolia and Cecropia peltata have been reported as hypoglycemic plants.
Among these plants Cecropia peltata L. was selected under the basis of our field work in
Yucatan, Mexico (3).

In our previous studies, the hypoglycemic effect of Cecropia obtusifolia was demonstrated
in stz-diabetic rats. The two main compounds (Cholorogenic acid, CA and Isoorientin, IO)
occurring on the active extracts were isolated (4). Also we have demonstrated the effect of
the aqueous extract in type 2 diabetic patients and correlated the hypoglycemic effect with
the presence of the previously isolated compounds (5). In a previous work (6) the
hypoglycemic effect of Cecropia obtusifolia and Cecropia pelatata were compared in a
non-diabetic animal model, which led to the conclusion that C. pelatata exerts as strong
hypoglycemic effect as C. obtusifolia. The authors related the effect to the Cholorogenic
acid content.

A possible mechanism of action of both Cecropias could be by reducing hepatic glucose

output, due to the inhibition of glucose 6 phosphatase by chlorogenic acid, which can
simultaneously target gluconeogenesis and glycogenolysis (2).

The n5-stz diabetic model is well recognized for the study of type 2 diabetes (7). A single
dose of STZ given to neonatal rats induces beta-cell injury, which is followed by limited
regeneration (short-term normalization of glycemia). At 6 to 15 weeks of age, an impaired
glucose disposal rate and significant beta-cell secretory dysfunction (type 2 diabetes) is
observed (7). We have already proved the suitability of this model for plant testing (8).

Cecropia peltata L. (Cecropiaceae), traditional Maya name “X cooch”, is a monopodic tree

15 m tall, growing as secondary vegetation in the tropical forest. It is highly appreciated in
the Mayan communities for the treatment of type 2 diabetes, this use was already
demonstrated by our group (9).

The aim of the present study was to investigate the hypoglycemic effect of the water and
butanol extracts of C. peltata in streptozotocin (n5-stz)-induced diabetic rats and compare
the phytochemical profile of the plant with the previously studied Cecropia obtusifolia (4).

Methods

Extracts Preparation.
For the phytochemical comparison of C. peltata with the previously studied compounds
from C. obtusifolia, the butanol (Be-P) extract was prepared from dry leaves as already
described (10). The leaves were collected near Chikinzonot Yucatan Mexico. A voucher
specimen was deposited at the Herbarium IMSS, 14696. After the first phytochemical
results more leaves were collected at two different locations near Playa del Carmen,
Quintana Roo México and Chetumal, Quinta Roo, Mexico. For C. obtusifolia a new
butanol extract (Be-O) was prepared from the already described plants (4, 5)

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The Be-P was used for the phytochemical analysis of the plant by HPLC as follows. An
extract sample was applied to a Nucleosil 60-30 C18 (Macherey & Nagel, Düren, Germany)
column and eluted with H2O/MeOH/AcCN 70:15:15, 4 ml/min monitored by DAD-HPLC,
Beckman System Gold with 32 Karat software. The Be-P was compared with the original
isolated samples from C. obtusifolia Be-O described in (4).
In addition, for the pharmacological testing, an aqueous extract (Ae-P) equivalent to the
traditional preparation was prepared from dry leaves as previously described (5). In our
previous studies (9, 4, 11) we had demonstrated that the butanol extract has the same
phytochemical composition as the water extract, but the active compounds are more
concentrated in the former.

Experimental animals and induction of type 2 diabetes.

Five-day old Wistar rats (weighing 10–12 g) received 90 mg/kg i. p. of STZ (Sigma, No.
242-646-8) in acetate buffer 0.1 M, pH 4.5. Control rats received only the buffer. After 4
weeks of age, rats were separated from their mothers and acclimatized with free access to
food and water in an air conditioned room (25° C with 55% humidity) under a 12:12 h
light: dark cycle, at the Bioterium of the Faculty of Sciences, UNAM. The animal handling
was in accordance with the Federal Government legislation of animal care. After 12 weeks,
diabetes was identified by measuring fasting plasma glucose levels. Animals with glucose
levels <155 mg/dl were not included in the study.

Experimental groups and Blood Collection.

The diabetic animals were classified into seven groups (2-8), each of them with eleven rats.
One non diabetic group (1) was included as control, which received 1.5 ml of physiological
NaCl-solution (Vehicle). Group 2 diabetic control received also 1.5 ml of physiological
NaCl-solution (vehicle), Group 3 were treated with the standard oral hypoglycemic agent
glibenclamide (3 mg/kg bodyweight (BW) in the same vehicle, Group 4 received a higher
dose of glibenclamide (5 mg/Kg), Groups 5 and 6 received Ae-P at 20 and 200 mg/kg bw,
respectively, Groups 7 and 8 received Be-P at 27 and 60 mg/kg bw, respectively. The
extracts were re-dissolved in 1.5 ml of physiological NaCl-solution and administered orally
with the use of a canule.

Blood samples were obtained from the tail vein (according to the Guideline 9 from IACUC,
3/10/99) before the oral administration of the extracts or the vehicle (T0), and at times 60,
120 and 180 min thereafter. Thirty-two microliters of blood were used for each assay.
Glucose concentration was measured in plasma with Reflotron equipment and confirmed
by Accutrend GC and Accu-check compact equipments (Roche).

Statistical analysis.
The data were statistically analyzed by one-way ANOVA followed by Tukey's test. Plasma
glucose concentrations are expressed as the mean ± standard error.

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Results
Phytochemistry.
The analysis of C. obtusifolia Be-O used in the present study showed that it contained both
CA and IO, in the same concentration as in the extract previously reported (ref; graphic 1).
However, after doing the same analysis with C. peltata Be-P from Chikinzonot, Yucatan,
we noticed that CA was not present in the extract (graphic 2). The analysis of leaf extracts
from the other two locations (Playa del Carmen and Chetumal, Quintana Roo, Mexico)
showed the same results (data not shown). The tree with the highest concentration of
metabolites in the phytochemical profile (graphic 3) was selected for the pharmacological
testing.
Hypoglycemic effect of C. pelatata.
The diabetic group (2) showed a significant hyperglycemia when compared against the
control group (1) since time 0. The two different doses of glibenclamide administered to the
diabetic animals from groups 3 and 4, exerted a hypoglycemic effect since time 60 up to the
end of the experiment (180 min). However group 4 (5 mg/kg) showed a higher
hypoglycemic effect at times 120 and 180 min than group 3 (3 mg/kg). The Ae-P showed a
hypoglycemic effect only at the higher dose at time 180 min (group 6), while group 5 did
not show effect. The Be-P at lower dose (group 7) showed effect only at 180 min., whereas
the higher dose showed an hypoglycemic effect since 60 min up to 180 min (Figure 1).

Graphic 1. UV-HPLC at 220 nm of Cecropia obtusifolia (green line) compared with

Cholorogenic acid (blue line) and Isoorientin (purple line).

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Graphic 2. UV-HPLC at 220 nm of Cecropia peltata (green line) compared with

Cholorogenic acid (blue line) and Isoorientin (purple line).

Graphic 3, Cecropia peltata DAD-HPLC spectra Upper left. Down right, 3d spectra .Upper
Right UV-Chromatogram Spectra at 220 nm (green line) compared with Cholorogenic acid
(blue line) and Isoorientin (purple line). Upper right Isoorientin from the present extract
compared with the extract previously published.

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Figure 1, Hypoglycemic effect of C. Peltata on n5-stz diabetic rats. G1, non diabetic
control; G2, diabetic control; G3, glibenclamide 3mg/kg; G4, glibenclamide 5 mg/kg; G5,
Ae-P 20 mg/kg; G6, Ae-P 200 mg/kg; G7, Be-P 27 mg/kg; G8, Be-P 60 mg/kg. * p<0.05,
** p<=.005 vs G2

Discussion

According to the present results we confirmed that the n5-stz rat is a suitable model for the
study of type 2 diabetes. Furthermore, we proved on this model that the hypoglycemic
effect produced by the oral administration of glibenclamide is dose dependent.
The hypoglycemic effect of Cecropia peltata, is not as clear as the previously observed
with C. obtusifolia (4), even when the here tested plant extracts were used at higher doses.
The water extract (Ae-P) exhibited a hypoglycemic effect only at the higher dose after 180
min. With the Be-P extract, the lower dose was as effective as the higher dose of the Ae-P,
and the higher dose of Be-P was the most effective, with the glycemia been significantly
lower earlier and decreasing with time. This seems to be due to the higher concentration of
compounds of Cecropia peltata reached in the Be-P.
In the here presented phytochemical results it can be observe that C. peltata contains lower
concentrations of cholorogenic acid (CA) and this can be the reason why at lower doses the

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extracts did not produce any hypoglycemic effect. Our results disagree with those of
Nicasio (6), who reported higher concentrations of CA. This difference could be due to a
variation on the CA content of C. peltata by phenotypic or seasonal variation. Since we
report the phytochemical analysis of several trees, we can assume that the content of
cholorogenic acid in C. peltata is in general lower than in C. obtusifolia.
With the here presented results, we can propose that the chlorogenic acid content in
Cecropia species plays an important role in their hypoglycemic effect. Similarly, we also
suggest that the isoorientin content can also be responsible of the strength of the
hypoglycemic effect.
The here presented results suggest that Cecropia species could be further developed as
phytomedicines and for observe a hypoglycemic effect it is necessary the presence of the
two main compounds occurring in the extracts.

Acknowledgments
To M.V. Z. Mario Soriano-Bautista, and Biol. Dora Salazar for housing the animals. This
work was partially supported by DGAPA, PAPIIT project IN202607 and CONACyT
special support to the first author.

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